(1) To develop animal, especially nonhuman primate, models that mimic human disease: In 2012-2013 two viruses emerged in the human population that caused severe respiratory disease with high case fatality rates: influenza A virus subtype H7N9 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). To better understand the pathogenicity of Influenza A virus H7N9, cynomolgus macaques were inoculated with A/Anhui/1/2013, a strain obtained from a fatal human case. Animals developed a transient, moderate pneumonia with virus replication occurring throughout the upper and lower respiratory tract. Clinical signs in the cynomolgus macaques were less severe than those observed in the majority of human cases of influenza A virus H7N9 infections. This likely reflects the importance of underlying medical conditions, observed in the majority of past and current human cases of H7N9 infection, for the development of severe or lethal infection with this virus (De Wit et al., in preparation). In attempt to develop an animal disease model for MERS-CoV that could be used for efficacy testing of countermeasures, we first inoculated Syrian hamsters with MERS-CoV. However, hamsters were not susceptible to infection showing no signs of disease nor seroconversion (de Wit et al., PLoS ONE 2013). Rhesus macaques inoculated with MERS-CoV developed a transient lower respiratory tract infection. Clinical signs, virus shedding, virus replication in respiratory tissues, gene expression and cytokine and chemokine profiles peaked early in infection and decreased over time. MERS-CoV caused a multifocal, mild to marked interstitial pneumonia, with virus replication occurring mainly in alveolar pneumocytes (Munster et al., New Engl J Med 2013). (2) To identify and characterize determinants of pathogenicity in animal models: To better understand the features that define highly lethal influenza viruses, we exploited a technology for the artificial generation and modification of influenza viruses (reverse genetics system). Using this technology, we have generated a consensus pandemic H1N1 virus (parent virus). In order to study virulence of the different strains and the mechanisms of pathogenesis we have generated selected recombinant viruses and characterized those mutants in vitro. For this we have compared the genome sequences of two early circulating pandemic H1N1 strains with different pathogenicity in humans (M4108, M4487). Sequence comparison revealed amino acid changes in 5 genome segments, PB2, PA, HA, NP and M. The recombinant viruses were generated on the background of M4108 by replacing the corresponding genomic segment. We have characterized the recombinant viruses in vitro and found preliminary evidence for a role of PB2 and PA. The in vivo studies are currently ongoing. The ferret model was attempted first, but the parent viruses did not significantly differ in disease outcome to make this model useful for our research question. We are currently investigating the use of a mouse model for this work (Tsuda et al, in preparation). Severe influenza virus infections are often associated with bacterial co-infections. This seems also be the case with the latest pandemic H1N1 virus. In order to study a potentiating effect of influenza and bacterial co-infection we performed a co-infection study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA only were largely asymptomatic, whereas animals infected with Ca04 only developed moderate pulmonary disease. Interestingly, animals initially infected with MRSA followed by Ca04 showed a dramatic reduction in clinical signs, whereas those initially infected with Ca04 followed by MRSA showed enhanced clinical disease (Kobayashi et al, in preparation). Similar studies were performed with a seasonal H3N2 virus and MRSA, in which we did not see disease reduction or enhancement (Kobayashi et al., Virulence: in revision). (3) To identify potential targets for intervention: In a first attempt to identify new targets for intervention we have performed a comparative transcriptomic analysis of acute host responses to infection with pandemic H1N1 (Ca04) in mice, macaques and swine. We observed differences in inflammatory molecules elicited, and the kinetics of their gene expression changes across these species, including genes regulated by nuclear receptor signaling molecules controlling lipid and metabolic processes impacting inflammatory responses to pandemic H1N1 influenza virus. While the heterodimeric retinoid X receptor (RXR) signaling pathway was differentially regulated during infection in each species, divergent expression of lipid-binding molecules and NF-kappaB molecules was observed across species. By comparing transcriptional changes across hosts in the context of clinical and virological measures and integrating evidence from transcription factor analyses, we were able to examine similarities and differences in acute host responses to pandemic H1N1 influenza infection across hosts (Go et al., BMC Genomics 2012). Targeted testing of potential antiviral agents against the newly emerged MERS-CoV is ongoing. This includes the identification of interferon-alpha2b and ribavirin as potentially effective antiviral agents against MERS-COV when used in combination in vitro (Falzarano et al., Sci Rep 2013). A follow-up study in the rhesus macaque model has indicated that combination treatment is effective at reducing clinical disease, evidence of pneumonia, pathology and inflammatory markers (both locally and systemically) (Falzarano et al., Nat Med: provisionally accepted 8/20/2013). In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV and efficacy testing in the rhesus macaque model is scheduled. Additional antiviral drugs have also been tested in vitro with nitazoxanide showing a selectivity of approximately 10. An efficacy assessment in rhesus macaques is planned pending delivery of pharmacokinetic data in the rhesus macaque. We have also recently received additional compounds to investigate in vitro including interferon-beta and the cyclophilin inhibitors DEB025 and NIM811. Results from in vitro studies are pending. (4) To develop vaccines and test their efficacy in the developed animal models: In order to develop a universal vaccine against influenza A viruses we are currently applying an approach based on two observations: i) highly conserved B cell epitopes are present within two separate helical regions within the hemagglutinin stalk region, and antibodies against them affords heterosubtypic binding and protection, and ii) removal of hemagglutinin globular region can increase antibody responses against other normally poorly antigenic epitopes. We used the Cytomegalovirus vector platform for these studies, which is able to induce long-lasting immune responses (both T cell and antibody) following a single vaccination. The vectors have been made and we are currently testing immunogenicity. The project title needs to be changed to better reflect the current and future work on this project. We would suggest changing as follows: Disease Modeling of Influenza and Other Emerging Respiratory Viral Pathogens.